Bottom Line:
Photobleaching leads to a depletion of fluorophores and a reduction of the brightness of protein complexes.We applied MSQ to measure the brightness of EGFP in E. coli and compared it to solution measurements.The results obtained demonstrate the feasibility of quantifying the stoichiometry of proteins by brightness analysis in a prokaryotic cell.

Affiliation: School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, United States of America.

ABSTRACTThe brightness measured by fluorescence fluctuation spectroscopy specifies the average stoichiometry of a labeled protein in a sample. Here we extended brightness analysis, which has been mainly applied in eukaryotic cells, to prokaryotic cells with E. coli serving as a model system. The small size of the E. coli cell introduces unique challenges for applying brightness analysis that are addressed in this work. Photobleaching leads to a depletion of fluorophores and a reduction of the brightness of protein complexes. In addition, the E. coli cell and the point spread function of the instrument only partially overlap, which influences intensity fluctuations. To address these challenges we developed MSQ analysis, which is based on the mean Q-value of segmented photon count data, and combined it with the analysis of axial scans through the E. coli cell. The MSQ method recovers brightness, concentration, and diffusion time of soluble proteins in E. coli. We applied MSQ to measure the brightness of EGFP in E. coli and compared it to solution measurements. We further used MSQ analysis to determine the oligomeric state of nuclear transport factor 2 labeled with EGFP expressed in E. coli cells. The results obtained demonstrate the feasibility of quantifying the stoichiometry of proteins by brightness analysis in a prokaryotic cell.

pone.0130063.g009: Measured stoichiometry of proteins in E. coli cells.MSQ-curves were fit to Eqs 14 and 22 to determine the average stoichiometry n for EGFP (triangles) and NTF2-EGFP (squares) as a function of the initial fluorescence intensity F0. The average stoichiometry of EGFP (gray dashed line) is 0.98 ± 0.10. The average stoichiometry of NTF2-EGFP (blue dashed line) is 1.94 ± 0.27. The top axis displays the initial protein concentration.

Mentions:
Now that we have a complete theory, we decided on the following strategy to analyze the MSQ-curve from an E. coli sample with unknown stoichiometry n. The experimental MSQ-curve is fit to Eqs 14 and 22 with n, kD, and τD as the only fit parameters. F0 is determined from a fit of the intensity decay curve, while N = TS/T, M = TDAQ/TS, and ΔfD = 1−exp(−kDTS) are functions of TS. The monomeric Q-factor Q1 of the function A is needed as a calibration factor and set equal to QEGFP,cyl to account for the geometry of the bacterium. Because the normalized brightness b and the stoichiometry n are numerically identical, b = n, we use both parameters interchangeably and at times refer to n as the normalized brightness. As a first test of this procedure we reanalyzed the FFS data from E. coli expressing EGFP with the new fit strategy to recover the stoichiometry of the sample. The analysis returned a normalized brightness n of ~1 for all samples (mean of 0.98 ± 0.10) as expected for a monomeric protein (Fig 9). The fit parameter kD varied slightly from cell to cell (mean 0.022 s-1 and standard deviation 0.0073 s-1), because of volume variations caused by different lengths of the E. coli cells. The diffusion time τD was approximately the same with a mean of 2.5 ± 0.9 ms.

pone.0130063.g009: Measured stoichiometry of proteins in E. coli cells.MSQ-curves were fit to Eqs 14 and 22 to determine the average stoichiometry n for EGFP (triangles) and NTF2-EGFP (squares) as a function of the initial fluorescence intensity F0. The average stoichiometry of EGFP (gray dashed line) is 0.98 ± 0.10. The average stoichiometry of NTF2-EGFP (blue dashed line) is 1.94 ± 0.27. The top axis displays the initial protein concentration.

Mentions:
Now that we have a complete theory, we decided on the following strategy to analyze the MSQ-curve from an E. coli sample with unknown stoichiometry n. The experimental MSQ-curve is fit to Eqs 14 and 22 with n, kD, and τD as the only fit parameters. F0 is determined from a fit of the intensity decay curve, while N = TS/T, M = TDAQ/TS, and ΔfD = 1−exp(−kDTS) are functions of TS. The monomeric Q-factor Q1 of the function A is needed as a calibration factor and set equal to QEGFP,cyl to account for the geometry of the bacterium. Because the normalized brightness b and the stoichiometry n are numerically identical, b = n, we use both parameters interchangeably and at times refer to n as the normalized brightness. As a first test of this procedure we reanalyzed the FFS data from E. coli expressing EGFP with the new fit strategy to recover the stoichiometry of the sample. The analysis returned a normalized brightness n of ~1 for all samples (mean of 0.98 ± 0.10) as expected for a monomeric protein (Fig 9). The fit parameter kD varied slightly from cell to cell (mean 0.022 s-1 and standard deviation 0.0073 s-1), because of volume variations caused by different lengths of the E. coli cells. The diffusion time τD was approximately the same with a mean of 2.5 ± 0.9 ms.

Bottom Line:
Photobleaching leads to a depletion of fluorophores and a reduction of the brightness of protein complexes.We applied MSQ to measure the brightness of EGFP in E. coli and compared it to solution measurements.The results obtained demonstrate the feasibility of quantifying the stoichiometry of proteins by brightness analysis in a prokaryotic cell.

Affiliation:
School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, United States of America.

ABSTRACTThe brightness measured by fluorescence fluctuation spectroscopy specifies the average stoichiometry of a labeled protein in a sample. Here we extended brightness analysis, which has been mainly applied in eukaryotic cells, to prokaryotic cells with E. coli serving as a model system. The small size of the E. coli cell introduces unique challenges for applying brightness analysis that are addressed in this work. Photobleaching leads to a depletion of fluorophores and a reduction of the brightness of protein complexes. In addition, the E. coli cell and the point spread function of the instrument only partially overlap, which influences intensity fluctuations. To address these challenges we developed MSQ analysis, which is based on the mean Q-value of segmented photon count data, and combined it with the analysis of axial scans through the E. coli cell. The MSQ method recovers brightness, concentration, and diffusion time of soluble proteins in E. coli. We applied MSQ to measure the brightness of EGFP in E. coli and compared it to solution measurements. We further used MSQ analysis to determine the oligomeric state of nuclear transport factor 2 labeled with EGFP expressed in E. coli cells. The results obtained demonstrate the feasibility of quantifying the stoichiometry of proteins by brightness analysis in a prokaryotic cell.